JP4452823B2 - Promoter with callus and seed embryo specific expression activity - Google Patents

Description

  The present invention relates to a promoter having callus and seed embryo-specific expression activity and use thereof.

  By introducing a gene in which a gene encoding a target protein to be expressed downstream of a promoter that can function in the plant cell is introduced into the plant cell, and regenerating the obtained plant cell by a normal plant tissue culture technique, An improved plant having a desired character has been produced. Examples of promoters commonly used in plants include cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium Ti plasmid-derived nopaline synthase gene promoter (Pnos), maize-derived ubiquitin promoter, rice-derived actin promoter, and the like. ing. These promoters are known to constitutively express a target protein in plant cells, and are widely used.

  However, when plants are improved, effective improvements may be achieved by, for example, local expression of the target protein, and as a part of developing new types of highly functional plants by such improvements, tissue specific It is desired to search for plant promoters that bring about specific expression.

  So far, as a promoter capable of tissue-specific expression of a target protein in a plant, rice is taken as an example, a rice cake-specific promoter RA8 (Patent Document 1), a rice cake or pollen-specific promoter CatA (Patent Document 2) ), Stamen-specific promoters T72, T23, T42, T155, E1 (patent document 3), flower organ-specific promoter RPC213 (patent document 4), mesophyll cell-specific promoter rbcS (non-patent document 1), endosperm-specific promoter GluB-1, GluA-2 (Non-patent Documents 2 and 3), callus-specific promoter PRO3 (Patent Document 5) and the like have been reported.

  On the other hand, in recent years, regarding the safety of genetically modified crops (GMO), antibiotic resistance genes such as kanamycin resistance gene and hygromycin resistance gene, or products of these genes (enzyme proteins) remain in the recombinant crops. It is regarded as a problem. This gene is called a selection marker gene for selecting cells in which the target gene has been integrated into the genome at an early stage, and is unnecessary after the plant body has been regenerated from the cells and further rooted and acclimatized. Therefore, it is preferable that the selection marker gene is expressed only at the time of selection of transformed cells and not expressed in various organs and tissues in the plant growth process, in consideration of plant growth, crop safety, and environmental impact. . However, when a constitutive promoter such as the CaMV 35S promoter is used, there is a problem that the selectable marker gene is expressed regardless of time or tissue.

Special table 2002-528125 International Publication WO00 / 58454 Special table hei 06-504910 International publication WO99 / 11800 JP2003-265182 Plant Physiol. 102, 991-1000 (1993) Plant Mol. Biol. 30, 1207-1221 (1996) Plant J. 4, 357-366 (1993)

  Accordingly, an object of the present invention is to provide a DNA having promoter activity that causes preferential expression in callus tissue, a recombinant plasmid containing the DNA and capable of preferentially expressing a selectable marker gene in callus, the set It aims at providing the transformed plant body which introduce | transduced the replacement vector.

  In order to solve the above problems, the present inventors refer to rice genome sequence information, rice EST appearance frequency information, rice full-length cDNA sequence information, etc. (http://www.dna.affrc.go.jp/). As a result of extensive research, we selected about 100 rice genes that are expected to have expression patterns specific to various tissues, and polymerase chain reaction (PCR) was performed on the 5 'upstream region of each selected gene. From the amplified fragments, the present inventors have succeeded in obtaining a DNA fragment having a promoter activity that preferentially expresses a target protein in callus, thereby completing the present invention.

That is, the present invention includes the following inventions.
(1) DNA which can function as a promoter, as shown in the following (a), (b) or (c).
(A) DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing
(B) a promoter activity consisting of a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 in the sequence listing, and causing specific expression in callus and seed embryos DNA
(C) DNA consisting of a part of the base sequence shown in SEQ ID NO: 1 in the sequence listing and having promoter activity that brings about specific expression in callus and seed embryo
(2) A DNA having a promoter activity that hybridizes with a DNA comprising a base sequence complementary to the DNA according to (1) under stringent conditions and brings about specific expression in callus and seed embryos.
(3) DNA obtained by linking a selection marker gene to the DNA described in (1) or (2).
(4) A recombinant vector containing the DNA according to (3).
(5) A recombinant vector comprising the DNA according to (3) and a gene encoding a target protein.
(6) A transformed plant into which the recombinant vector according to (4) or (5) has been introduced.
(7) The recombinant vector according to (4) or (5) is introduced into a plant material, the plant material is cultured in a callus-forming medium, and the presence or absence of expression of a selection marker gene from the cultured callus is an indicator. A method for selecting a transformed plant body, comprising selecting a transformed callus.

  According to the present invention, DNA having promoter activity that provides specific expression to callus and seed embryos is provided. By using this DNA having promoter activity, a selective marker gene such as an antibiotic resistance gene is preferentially expressed in callus cells, and in other tissues or organs such as edible portions where the selection marker gene product does not remain favorably. It can suppress the expression in the leaves and endosperm of a certain plant, and does not affect the growth of the plant. Therefore, it is useful for production of highly safe genetically modified plants.

1. Promoter and Isolation thereof DNA capable of functioning as a promoter according to the present invention (hereinafter referred to as “promoter”) is a DNA comprising the base sequence shown in SEQ ID NO: 1.

  The DNA of the present invention induces the expression of the selection marker gene in callus by inserting it into the 5 ′ side of the selection marker gene for efficiently selecting transformed plant cells, or It can be expressed at high levels in callus.

  Examples of the selection marker gene include a hygromycin resistance gene, a kanamycin resistance gene, a bialaphos resistance gene, a blasticidin S resistance gene, an acetolactate synthase gene, and the like.

  In addition, the DNA of the present invention comprises a promoter comprising a nucleotide sequence in which one or more bases are substituted, deleted, added or inserted in the nucleotide sequence shown in SEQ ID NO: 1 and which causes specific expression in callus and seed embryos Active DNA is also included. Here, the number of bases that may be substituted, deleted, added or inserted is not particularly limited, but is preferably 1 to several. For example, 1 to 10, preferably 1 to 5 bases of the nucleotide sequence shown in SEQ ID NO: 1 may be deleted, and 1 to 10, preferably 1 to 5 nucleotide sequences shown in SEQ ID NO: 1. May be added, or 1 to 10, preferably 1 to 5 bases of the base sequence shown in SEQ ID NO: 1 may be substituted with other bases.

  Furthermore, the DNA of the present invention includes a DNA consisting of a part of the base sequence shown in SEQ ID NO: 1 and having promoter activity that brings about specific expression in callus and seed embryos.

  The essential part of the promoter activity in the DNA consisting of the base sequence shown in SEQ ID NO: 1 is a variety of deletions of the DNA, for example, DNA fragments deficient in various lengths from the 5 ′ upstream side to beta-glucuronidase (GUS ) It can be identified by introducing a plasmid fused with a reporter gene such as a gene into a host and measuring the promoter activity. Techniques for identifying such an active moiety are known to those skilled in the art.

  Such mutant DNAs only need to have a promoter activity (hereinafter referred to as “callus and seed embryo-specific promoter activity”) that causes expression specific to callus and seed embryo, and the magnitude of the activity is as follows. Although not particularly limited, it is preferable that the callus and seed embryo-specific promoter activity of the DNA consisting of the base sequence shown in SEQ ID NO: 1 is substantially retained. “Substantially retaining the callus and seed embryo-specific promoter activity of the DNA comprising the base sequence shown in SEQ ID NO: 1” means that, in the actual usage mode using the promoter activity, the base sequence shown in SEQ ID NO: 1 The activity is maintained to the extent that it can be used in the same conditions as DNA.

  In the present invention, “callus and seed embryo-specific promoter activity” refers to an activity that preferentially and highly expresses a target gene in callus and seed embryo over at least one other tissue or organ of the same plant. Say.

  The introduction of gene mutation for obtaining mutant DNA as described above can be carried out by a known technique such as the Kunkel method or the Gapped duplex method or a method analogous thereto, for example, mutation using site-directed mutagenesis. Kits for introduction (for example, Mutant-K (manufactured by TAKARA) or Mutant-G (manufactured by TAKARA)) and LA PCR in vitro Mutagenesis series kits by TAKARA can be used.

  Furthermore, those skilled in the art can use DNA consisting of all or part of the base sequence shown in SEQ ID NO: 1 to perform the same function as DNA consisting of the base sequence shown in SEQ ID NO: 1, ie, callus and seed embryo-specific. It is also easy to newly obtain and use DNA consisting of other base sequences having promoter activity from various organisms. For obtaining such a DNA consisting of another base sequence, for example, it is hybridized under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of all or part of the base sequence shown in SEQ ID NO: 1. It can be performed by hybridization, PCR using a part of the base sequence as a primer, or the like. Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, a highly homologous nucleic acid, that is, a complementary strand of DNA consisting of a nucleotide sequence having a homology of 90% or more, preferably 95% or more, with the nucleotide sequence shown in SEQ ID NO: 1 hybridizes and has a lower homology. A condition in which the complementary strand of the nucleic acid does not hybridize is mentioned. More specifically, the sodium concentration is 10 to 300 mM, preferably 15 to 75 mM, and the temperature is 25 ° C to 70 ° C, preferably 42 ° C to 55 ° C.

Whether the mutant DNA obtained as described above or the homolog obtained by hybridization has activity as a promoter is determined by various reporter genes such as beta-glucuronidase (GUS), luciferase (LUC), green fluorescent protein (GFP ),
A vector in which genes such as chloramphenicol acetyltransferase (CAT), beta galactosidase (GAL), nopaline synthase (NOS), octopine synthase (OCS) and the like are linked to the downstream region of the above promoter is prepared, and the vector is used. It can be confirmed by measuring the expression of the reporter gene after insertion into the genome of a plant cell by various transformation methods (to be described later) that have been well known and conventionally used.

  For example, when the reporter gene is GUS, the promoter activity in the host cell is determined by (i) a method by histochemical GUS staining (EMBO J. 6, 3901-3907 (1987)) and / or (ii) ) GUS activity was measured according to the Castle & Morris method (Plant Molecular Biology Manual, B5, 1-16 (1994); SBGelvin & RASchilperoort, Kluwer Academic Publishers) using a fluorescent substrate, and Bradford's method (Anal. Biochem. 72). , 248-254 (1976)), and the GUS activity can be confirmed by converting the GUS activity per protein amount (calculated as nmole 4-MU / min / mg protein).

  The promoter of the present invention is present on the chromosome 8 genome of rice (Nipponbare), and the rice gene encoding a 33 kDa secreted protein having the amino acid sequence shown in SEQ ID NO: 3 [DDBJ (http: //www.ddbj. nig.ac.jp): Acquired by isolating the upstream region of the gene (hereinafter referred to as “rice # 15 gene”) based on the base sequence (SEQ ID NO: 2) of Accession No. AK073888] it can. The method for isolating the promoter region is not particularly limited, and examples thereof include inverse PCR, a method for isolating from a genomic DNA library, and the like.

  In the case of inverse PCR, a pair of primers is synthesized based on the nucleotide sequence information of the rice # 15 gene, PCR is performed using these paired primers and a genomic DNA fragment that has been treated with a predetermined restriction enzyme and then self-ligated. By doing so, the upstream region of the rice # 15 gene can be amplified. Thereafter, by cloning the upstream region of the rice # 15 gene and determining the nucleotide sequence, the promoter region of the rice # 15 gene can be isolated and the nucleotide sequence (SEQ ID NO: 1) can be determined.

  In the case of isolation from a genomic DNA library, genomic DNA containing rice # 15 gene is screened from a genomic DNA library prepared according to a conventional method using cDNA containing rice # 15 gene as a probe. Then, by determining the nucleotide sequence of the screened genomic DNA, the promoter region present in the upstream region of the rice # 15 gene can be identified, and further, only the promoter region is amplified and cloned by PCR or the like. It can be isolated.

  Once the base sequence of the promoter of the present invention has been determined, the subsequent synthesis is carried out by chemical synthesis or by using a DNA consisting of a part of the base sequence as a primer, and using the rice total DNA as a template. It can be easily obtained by PCR.

  Further, by measuring the promoter activity using a part of the isolated promoter region (SEQ ID NO: 1), the region contributing to the promoter activity in the isolated promoter region (SEQ ID NO: 1) is specified. Can do. A part of the promoter region can be obtained by appropriately using a method in which a part of the promoter region is amplified by PCR, a method in which the promoter region is treated with a predetermined restriction enzyme, and fragmented.

  A part of the obtained promoter region can be incorporated upstream of a gene whose expression level can be quantified, and the promoter activity can be measured by quantifying the expression level of the gene. That is, it can be obtained by constructing a recombinant vector incorporating a part of the obtained promoter region and a predetermined gene, and quantifying the expression level of the gene in cells transformed with the recombinant vector. Promoter activity in a part of the promoter region can be measured.

2. Recombinant vector The recombinant vector of the present invention comprises the above-mentioned 1. It can be constructed by ligating a DNA in which a selection marker gene is ligated to the above promoter to an appropriate vector. Here, as a vector, a target gene can be introduced into a plant via Agrobacterium, pBI system, pPZP system (Hajdukiewicz P, Svab Z, Maliga P .: The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation., Plant Mol Biol., 25: 989-94, 1994), pCAMBIA (http://www.cambia.org/main/r_et_camvec.htm), pSMA vectors, and the like are preferably used. In particular, pBI binary vectors or intermediate vector systems are preferably used, and examples thereof include pBI121, pBI101, pBI101.2, pBI101.3 and the like. A binary vector is a shuttle vector that can replicate in Escherichia coli and Agrobacterium. When a plant is infected with Agrobacterium that holds the binary vector, it is a border sequence consisting of LB and RB sequences on the vector. It is possible to incorporate the enclosed DNA into plant nuclear DNA (EMBO Journal, 10 (3), 697-704 (1991)). On the other hand, a pUC vector can directly introduce a gene into a plant, and examples thereof include pUC18, pUC19, and pUC9. Plant virus vectors such as calflower mosaic virus (CaMV), kidney bean mosaic virus (BGMV), and tobacco mosaic virus (TMV) can also be used.

  To insert a gene encoding a target protein (hereinafter referred to as a target gene) into a vector, first, the purified DNA is cleaved with a suitable restriction enzyme and inserted into a restriction vector site or a multicloning site of a suitable vector DNA. Then, a method of linking to a vector is employed.

  The target gene is not particularly limited, and has antibacterial properties against specific bacteria, ability to synthesize specific useful substances, sensitivity to specific plant hormones, or morphological characteristics different from the original plant (eg, hatching, elongation promotion, enlargement, etc.) ) And the like.

  The target gene described above needs to be incorporated into a vector so that the function of the gene is exhibited. Therefore, in the vector, the above 1. above, inside, or downstream of the target gene. A second promoter, an enhancer, an intron, a poly A addition signal, a 5′-UTR sequence, etc., which are different from the above can be linked.

  As the second promoter, any DNA can be used as long as it functions in plant cells and can induce expression in a specific tissue of a plant, in a specific developmental stage, or by a specific environmental factor or stimulus. It does not have to be derived. Specific examples include a cauliflower mosaic virus (CaMV) 35S promoter, a nopaline synthase gene promoter (Pnos), a corn-derived ubiquitin promoter, a rice-derived actin promoter, a tobacco-derived PR protein promoter, and the like.

  Examples of the enhancer include an enhancer region that is used to increase the expression efficiency of the target gene and includes an upstream sequence in the CaMV35S promoter.

  The terminator may be any sequence that can terminate transcription of the gene transcribed by the promoter, and examples thereof include nopaline synthase gene terminator, octopine synthase gene terminator, CaMV 35S RNA gene terminator, and the like.

  Alternatively, the selection marker gene may be prepared by ligating the same gene together with the target gene as described above to prepare a recombinant vector, or alternatively, a recombinant vector obtained by linking the selection marker gene to a plasmid, A recombinant vector obtained by ligating the target gene to a plasmid may be prepared separately. When prepared separately, each vector is co-transfected (co-introduced) into the host.

3. Transformed plant body 2. Using the recombinant vector prepared in Step 1, a target plant can be transformed to prepare a transformed plant body.

  In preparing a transformed plant body, various methods already reported and established can be used as appropriate. Preferred examples thereof include the Agrobacterium method, the PEG-calcium phosphate method, the electroporation method, Examples include the liposome method, particle gun method, and microinjection method. When the Agrobacterium method is used, a protoplast may be used or a tissue piece may be used. When using protoplasts, co-culture with Agrobacterium with Ti plasmid, fusing with spheroplasted Agrobacterium (spheroplast method), and when using tissue fragments, aseptic culture of the target plant It can be carried out by infecting leaf pieces (leaf discs) or infecting callus.

  Whether or not a gene has been incorporated into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, Western blotting, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing a DNA-specific primer. After PCR, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band. By doing so, it can confirm that it transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, the amplification product may be bound to a solid phase such as a microplate, and the amplification product may be confirmed by fluorescence or enzymatic reaction.

  Plants used for transformation in the present invention include monocotyledonous plants such as rice, wheat, corn, leek, lily, orchid, soybean, rapeseed, tomato, potato, chrysanthemum, rose, carnation, petunia, gypsophila, cyclamen and the like. Plants such as dicotyledonous plants can be mentioned, and are not particularly limited. Preferably, plants of the family Gramineae from which the DNA of the present invention has been isolated, for example, plants such as rice, barley, wheat, corn, sugarcane, buckwheat, sorghum, millet and millet.

  In the present invention, examples of plant materials to be transformed include roots, stems, leaves, seeds, embryos, ovules, ovaries, shoot tips (plant growth points at the tips of buds), cocoons, pollen and the like. Plant tissue and its sections, undifferentiated callus, and plant cultured cells such as proplast from which the cell wall is removed by enzyme treatment can be mentioned. In selecting a transformed plant body at the callus stage (see below 4). )), And preferably a plant material that can be dedifferentiated into callus or callus.

  In the present invention, the transformed plant body means the whole plant body, plant organ (eg, root, stem, leaf, petal, seed, seed, fruit, etc.) plant tissue (eg, epidermis, phloem, soft tissue, xylem, Vascular bundles, etc.) and plant cultured cells.

  When plant cultured cells are targeted, in order to regenerate a transformant from the obtained transformed cells, an organ or an individual may be regenerated by a known tissue culture method. Such an operation can be easily performed by those skilled in the art by a method generally known as a method for regenerating plant cells from plant cells. Regeneration from plant cells to plants can be performed, for example, as follows.

  First, when plant materials or protoplasts are used as plant materials to be transformed, these are inorganic elements, vitamins, carbon sources, sugars as energy sources, plant growth regulators (plant hormones such as auxin and cytokinin) And so on, and cultured in a sterilized callus formation medium to form dedifferentiated callus that grows irregularly (hereinafter referred to as “callus induction”). The callus thus formed is transferred to a new medium containing a plant growth regulator such as auxin, and further grown (subcultured).

  When callus induction is performed in a solid medium such as agar and subculture is performed in, for example, liquid culture, each culture can be performed efficiently and in large quantities. Next, callus grown by the above subculture is cultured under appropriate conditions to induce organ redifferentiation (hereinafter referred to as “redifferentiation induction”), and finally regenerates a complete plant body. . Redifferentiation induction can be performed by appropriately setting the types and amounts of various components such as plant growth regulators such as auxin and cytokinin, carbon sources, and the like, light, temperature and the like in the medium. By such redifferentiation induction, somatic embryos, adventitious roots, adventitious shoots, adventitious foliage, etc. are formed and further grown into complete plants. Alternatively, storage or the like may be performed in a state before becoming a complete plant body (for example, encapsulated artificial seeds, dried embryos, freeze-dried cells and tissues).

  The transformed plant of the present invention is not only the “T1 generation” that is the regenerated generation that has undergone transformation, but also the “T2 generation” that is a progeny obtained from the seeds of the plant, analysis by drug selection or Southern method, etc. It includes progeny plants such as the next generation (T3 generation) obtained by self-pollination of "T2 generation" plants that have been found to be transgenic.

4). Transformed plant body selection marker and selection method The promoter of the present invention expresses the selection marker gene only in callus and seed embryos and not in other tissues such as roots, stems, leaves, etc. The ligated DNA can be used as a novel selection marker that can be expressed only when selecting transformed cells. In addition, in rice incorporating a marker gene that is subject to expression control of the promoter, expression of the marker gene is also observed in seed embryos other than callus, but since the harvested rice seeds are removed by rice milling, the embryo part is removed, The endosperm part where the marker gene expression is not observed can be an edible part.

  That is, according to the present invention, a recombinant vector containing a DNA having a selection marker gene linked to the above promoter sequence alone or together with other target genes to be expressed is constructed, and this is introduced into plant material to introduce callus. There is provided a method for selecting a transformed plant comprising culturing in a forming medium and selecting a transformed callus from the cultured callus using the presence or absence of expression of a selection marker gene as an index.

  For example, if the selection marker gene is an antibiotic resistance gene such as the kanamycin resistance gene, callus induced by culturing plant material introduced with a recombinant vector in a callus formation medium is used. It can be judged by further culturing in a medium containing the corresponding antibiotic and examining the presence or absence of antibiotic resistance from the cultured callus.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.
(Example 1) Isolation of rice # 15 gene promoter sequence and introduction into transformation vector (1) Isolation of promoter
Isolation of a promoter sequence located 5 ′ upstream of the rice # 15 gene (DDBJ (http://www.ddbj.nig.ac.jp): accession number AK073888) having the base sequence shown in SEQ ID NO: 2 This was performed by genomic PCR. DDBJ (http://www.ddbj.nig.ac.jp: Accession number) Using genomic DNA extracted from green leaves of rice (variety: Nipponbare) seedlings using DNeasy Plant Mini Kit (Qiagen) as a template AP005620) Primer # 15-5 (5'-ATGC AAGCTT CTCTCACTGGTCACTTGTATG-3 ': SEQ ID NO: 4) and # 15-3 (5'-GCCG CCATGG ATGGCGCCAATTATGTACTC-3: Sequence designed from rice genome sequence information registered in AP005620) PCR was performed using No. 5) as a primer. The primer is 1871 from the base of 1865 base pairs [position 7 in SEQ ID NO: 1 (positions 1 to 6 are HindIII recognition sequences) located 5 ′ upstream of the translation initiation codon ATG predicted from the mRNA information of the # 15 gene. It was designed to amplify the sequence (positions 1870 to 1871 correspond to bases up to the NcoI recognition sequence cc ATG g). In this 1865 base pair sequence, there is a TATA sequence 156 bp upstream from the initiation codon, and a CCAAT box that is thought to be involved in transcriptional activation 170 bp upstream. The primers also introduce unique HindIII and NcoI restriction enzyme recognition sites at the 5 'end (# 15-5) and 3' end (# 15-3), respectively, to facilitate cloning of the amplified fragment. did.

  After PCR, the amplified fragment (approx. 1.9 kb) is treated with Ex Taq polymerase (Takara) in the presence of dATP at 72 ° C for 5 minutes to add A to the end, and using Promega's pGEM-T easy vector system And cloned into the pGEM-T easy vector.

After cloning, the base sequence of the insert was decoded and confirmed to be the same as the above registered rice genome sequence information. SEQ ID NO: 1 shows the base sequence. In the nucleotide sequence of SEQ ID NO: 1, the nucleotide sequence from position 1 to position 6, the nucleotide sequence from position 1870 to position 1875 is the restriction enzyme site used for cloning, and the nucleotide sequence from position 1746 is the transcription region. .
The cloning procedure of the rice gene promoter sequence described above is shown in FIG.

(2) Construction of Binary Ti Plasmid Vector for Plant Transformation In order to introduce the above promoter sequence into rice, a binary Ti plasmid vector pSMAHdN627-M2GUS for plant transformation was constructed (FIG. 2). As shown in FIG. 2, this binary vector has an Agrobacterium Ti plasmid-derived Nos (nopaline synthase gene) promoter :: E. coli-derived HPT (hygromachine resistance) gene as a plant selection marker gene on T-DNA. The coding region of :: Ti plasmid-derived TiaaM (tryptophan monooxygenase gene) terminator was placed so that the transformed plant body was resistant to hygromycin B. In addition, a chimeric gene composed of a beta glucuronidase (GUS) gene coding region :: Ti plasmid-derived Nos terminator is placed in the adjacent site, and a multicloning site is inserted so that a promoter sequence can be inserted 5 'upstream of the GUS gene. Was provided. Also, outside of the left border (LB) and right border (RB) sequences, a spectinomycin resistance (Sp ^R ) gene that can function in microbial cells, a pBR322-derived (ColE1 type) replication initiation region that functions in E. coli The sequence Sta for stably maintaining the plasmid in Agrobacterium cells and the replication initiation region Rep were provided.

(3) Introduction of promoter sequence into binary vector
The rice # 15 gene promoter fragment was excised by double digestion of pGEM-T easy vector with HindIII and NcoI, and cloned into the corresponding site of the pSMAHdN627-M2GUS vector constructed in (2). The obtained plasmid was designated as pSMAHdN627-15GUS, and electroporation method (0.2 cm cuvette, pulse conditions: 2.4 kV / cm, 25 μF, 200Ω) using E. coli pulsar manufactured by Biorad Co. was used. It was introduced into the system.

(Example 2) Rice transformation Rice was transformed by an ultra-rapid transformation method (see WO 01/06844 A1 (2001)). Rice (variety: Nipponbare) seeds are sterilized with 70% ethanol followed by sodium hypochlorite, rinsed with sterile distilled water, drained, and N6D agar medium [N6 salts and vitamins so that the embryos face up (Chu CC, CSWang, CCSun, C. Hsu, KC Yin, CY Chu, Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources, Sci. Sinica, 18, 659-668 (1975)), 30 g / L sucrose, 0.3 g / L casamino acid, 2.8 g / L proline, 2 mg / L 2,4-D, 2 g / L gellite, pH 5.7) at 30 ° C under bright conditions For 5 days.

  On the other hand, Agrobacterium EHA105 strain carrying plasmid pSMAHdN627-15GUS inserted with rice # 15 gene promoter fragment contains 25 mg / L chloramphenicol, 25 mg / L rifampicin, and 100 mg / L spectinomycin The cells were cultured on LB agar medium at 28 ° C. for 3 days. Subsequently, the grown cells are scraped in a small amount with microspatel, and AAM liquid medium containing 20 mg / L acetosyringone [Hiei, Y., Ohta, S., Komari, T., Kumashiro, T., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J., 6, 271-282 (1994)]. Rice seeds cultured for 5 days in N6D medium were immersed in this Agrobacterium suspension, and the bacterial solution was thoroughly cut off, then placed on an AAM agar medium containing 20 mg / L acetosyringone and darkened at 25 ° C. Cultivation was performed under conditions for 3 days (co-culture). Wash the rice germinated seeds after co-cultivation with sterilized water, followed by sterilized water containing 500 mg / L carbenicillin. It was placed on an N6D agar medium containing 30 mg / L hygromycin B and cultured for 14 days under light conditions at 30 ° C. Thereafter, the grown shoot base was transformed into a recombinant redifferentiation and selective medium [plant regeneration medium: MS salts and vitamins (Murashige, T., Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue. cultures, Physiol. Plant, 15, 473-497 (1962)), 30 g / l sucrose, 30 g / l sorbitol, 2 g / l casamino acid, 0.02 mg / l NAA, 2 mg / l kinetin, 2 g / l Gelrite, pH 5.8] with 300 mg / L carbenicillin + 30 mg / L hygromycin B added; Toki, S., 1997, Rapid and Efficient Agrobacterium-mediated transformation of rice, Plant Mol Biol. Rep. , 15: 16-21], and cultured for 14 days at 30 ° C. and in light conditions, and this operation was repeated once more to redifferentiate plants having hygromycin resistance. The redifferentiated individuals thus obtained were transplanted to MS hormone-free agar medium [MS salts and vitamins (Murashige, T. et al., Supra), 30 g / l sucrose, 4 g / l gellite, pH 5.8]. It was grown for 1 to 2 weeks at 30 ° C. under light conditions to promote the elongation of the shoot (aboveground part) and the rooting and elongation. When the length of the shoots and roots of the transformants reached about 10 cm or more in a petri dish (9 cm in diameter), they were transplanted to the culture soil and clarified in the growth chamber for growing the recombinants. Further growth was carried out in a cycle of 14 hours (30 ° C.)-dark 10 hours (25 ° C.).

(Example 3) Preparation of plant tissue section and observation of transgene expression by GUS staining In order to observe the activity of the # 15 gene promoter introduced into rice, histochemical staining of GUS enzyme activity was performed. Of the plant tissue materials used in the experiment, callus, flower organs, and roots were directly immersed in the reaction solution after being cut from rice individuals or callus (cultured cells). For leaf blades, the developed leaves were cut to a width of 5-10 mm, embedded in 5% agar, and 80 μm-thick sections were prepared using a microslicer (Dosaka EM, DTK1000). 4 281-285 (1992)]. For the heel base, cut 5-10 mm of tissue from the root of the cut potato (cane: a term representing the stem of the grass family), and for the shoot apex, the portion immediately above the heel base is 10 mm. A section having a thickness of 80 μm was prepared in the same manner as the blade. The plant material thus prepared was used as a reaction solution for measuring GUS activity (50 mM sodium phosphate buffer (pH 7.0), 1 mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 5% (v / v) methanol, 10 μg / ml cycloheximide, 1 mM dithiothreitol] and placed at 37 ° C. with gentle shaking for 24 hours. Thereafter, the reaction solution was replaced with 100% ethanol to stop the reaction, and the mixture was further allowed to stand for 24 hours to remove chlorophyll from the cells of the green tissue. Next, after replacing ethanol with pure water, the staining pattern was observed with a stereomicroscope or an optical microscope. As a result, a blue color indicating the expression of the GUS gene in callus cells was particularly strongly detected. In addition, relatively strong activity appeared in the embryonic tissue of T2 progeny seeds obtained by breeding redifferentiated individuals (T1 generation). In other tissues and organs, no GUS expression was observed except for slight GUS expression in the vascular bundle at the base of the eyelid (FIG. 3).

  Further, progeny seeds (T2 generation) harvested from regenerated different generation (T1) rice individuals (1 line) were cultured on the N6D agar medium of Example 2 at 30 ° C. for 14 days to form callus. When the callus was immersed in a GUS activity reaction solution and placed at 37 ° C. for 24 hours, GUS activity was observed in callus formed from 8 of 9 grains. This suggests that the # 15 promoter :: GUS DNA fragment is inserted in the transforming generation (T1 generation) rice genome in two to several copies and is stably transmitted to the next generation (T2 generation). It was done.

The cloning procedure of the rice gene promoter of this invention is shown. The structure of a binary Ti plasmid vector for plant transformation: pSMAHdN627-M2GUS is shown. The GUS dyeing | staining result in each plant material is shown.

Claims (6)

DNA which can function as a promoter, shown in the following (a), (b) or (c).
(A) DNA consisting of the base sequence shown in SEQ ID NO: 1 in the sequence listing
(B) Promoter activity that consists of a base sequence in which 1 to 10 bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 in the sequence listing and brings about specific expression in callus and seed embryos DNA
(C) DNA comprising a nucleotide sequence having a homology of 95% or more with respect to the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, and having a promoter activity that brings about specific expression in callus and seed embryos. DNA comprising a selection marker gene linked to the DNA of claim 1 . A recombinant vector containing the DNA according to claim 2 . A recombinant vector containing the DNA of claim 2 and a gene encoding the target protein. A transformed plant into which the recombinant vector according to claim 3 or 4 is introduced. A recombinant vector according to claim 3 or 4 is introduced into plant material, the plant material is cultured in a callus forming medium, and transformed callus is expressed using the presence or absence of expression of a selection marker gene from the cultured callus as an indicator. A method for selecting transformed plants, comprising selecting.

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